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Raquel Barbuio, Marciane Milanski, Manoel B Bertolo, Mário J Saad and Lício A Velloso

Non-alcoholic fatty liver disease, induced by nutritional factors, is one of the leading causes of hepatic dysfunction in the modern world. The activation of proinflammatory signaling in the liver, which is induced by systemic and locally produced cytokines, and the development of hepatic insulin resistance are two important factors associated with the progression from steatosis to steatohepatitis, a pre-cirrhotic condition. The objective of the present study was to evaluate the effect of inhibition of tumour necrosis factor (TNF)-α , using the monoclonal antibody infliximab, on the expression of cytokines, induction of steatosis and fibrosis, and insulin signal transduction in the liver of Wistar rats fed a high-fat diet. Ten days of treatment with infliximab significantly reduced the expression of the proinflammatory markers, TNF-α , IL-6, IL-1β , and SOCS-3, in the liver of rats fed a high-fat diet. This was accompanied by reduced fat deposition and fibrosis and by improved insulin signal transduction through insulin receptor (IR)/IR substrate/Akt/FOXO1 and JAK2/STAT3 pathways. In conclusion, short-term inhibition of TNF-α with infliximab reduces inflammation and steatosis/fibrosis, while improving insulin signal transduction in an animal model treated with a high-fat diet.

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Márcio Pereira-da-Silva, Cláudio T De Souza, Alessandra L Gasparetti, Mário J A Saad and Lício A Velloso

Transgenic hyperexpression of melanin-concentrating hormone (MCH) produces a phenotype of obesity and glucose intolerance. However, it is not known whether under this specific condition, glucose intolerance develops as a direct consequence of hyperexpressed MCH or is secondary to increased adiposity. Here, rats were treated i.c.v. with MCH or with an antisense oligonucleotide to MCH (MCH-ASO). MCH promoted an increase in blood glucose and a decrease in blood insulin levels during a glucose tolerance test. MCH also caused a decrease in the constant of glucose disappearance during an insulin tolerance test. All these effects of MCH were independent of body weight variation and were accompanied by reduced insulin receptor substrate (IRS)-1 engagement of phosphatidylinositol-3 kinase (PI3-kinase) in white and brown adipose tissues, skeletal muscle and liver and by reduced Akt activation in skeletal muscle. MCH also led to a significant reduction in ERK activation in white adipose tissue. Finally, inhibition of hypothalamic MCH expression promoted a significant increase in ERK activation in brown adipose tissue. We conclude that hypothalamic MCH controls glucose homeostasis through mechanisms that are, at least in part, independent of adiposity.

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Daniela F Bertelli, Andressa Coope, Andrea M Caricilli, Patricia O Prada, Mario J Saad, Licio A Velloso and Eliana P Araujo

The 72 kDa inositol polyphosphate 5-phosphatase E (72k-5ptase) controls signal transduction through the catalytic dephosphorylation of the 5-position of membrane-bound phosphoinositides. The reduction of 72k-5ptase expression in the hypothalamus results in improved hypothalamic insulin signal transduction and reduction of food intake and body mass. Here, we evaluated the tissue distribution and the impact of obesity on the expression of 72k-5ptase in peripheral tissues of experimental animals. In addition, insulin signal transduction and action were determined in an animal model of obesity and insulin resistance treated with an antisense (AS) oligonucleotide that reduces 72k-5ptase expression. In lean Wistar rats, 72k-5ptase mRNA and protein are found in highest levels in heart, skeletal muscle, and white adipose tissue. In three distinct models of obesity, Wistar rats, Swiss mice fed on high-fat diet, and leptin-deficient ob/ob mice, the expression of 72k-5ptase is increased in skeletal muscle and adipose tissue. The treatment of obese Wistar rats with an anti-72k-5ptase AS oligonucleotide results in significant reduction of 72k-5ptase catalytic activity, which is accompanied by reduced food intake and body mass and improved insulin signal transduction and action as determined by immunoblotting and clamp studies respectively. 72k-5ptase expression is increased in obesity and its AS inhibition resulted in a significant improvement in insulin signal transduction and restoration of glucose homeostasis.

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Andréa M Caricilli, Paula H Nascimento, José R Pauli, Daniela M L Tsukumo, Lício A Velloso, José B Carvalheira and Mário J A Saad

The aims of the present study were to investigate the expression of toll-like receptor 2 (TLR2) in muscle and white adipose tissue (WAT) of diet-induced obesity (DIO) mice, and also the effects of its inhibition, with the use of TLR2 antisense oligonucleotide (ASON), on insulin sensitivity and signaling. The expression of TLR2 was increased in muscle and WAT of DIO mice, compared with those that received standard chow. Inhibition of TLR2 in DIO mice, by TLR2 ASON, improved insulin sensitivity and signaling in muscle and WAT. In addition, data show that the inhibition of TLR2 expression prevents the activation of IKBKB, MAPK8, and serine phosphorylation of IRS1 in DIO mice, suggesting that TLR2 is a key modulator of the crosstalk between inflammatory and metabolic pathways. We, therefore, suggest that a selective interference with TLR2 presents an attractive opportunity for the treatment of insulin resistance in obesity and type 2 diabetes.

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Graziela R Stoppa, Maristela Cesquini, Erika A Roman, Patrícia O Prada, Adriana S Torsoni, Talita Romanatto, Mario J Saad, Licio A Velloso and Marcio A Torsoni

We hypothesized that citrate might modulate the AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK)/(ACC) pathway and participate in neuronal feeding control and glucose homeostasis. To address this issue, we injected citrate into the lateral ventricle of rats. Intracerebroventricular (ICV) injection of citrate diminished the phosphorylation of hypothalamic AMPK/ACC, increased the expression of anorexigenic neuropeptide (pro-opiomelanocortin and corticotropin-releasing hormone), elevated the level of malonyl-CoA in the hypothalamus, and reduced food intake. No change was observed in the concentration of blood insulin after the injection of citrate. With a euglycemic–hyperinsulinemic clamp, the glucose infusion rate was higher in the citrate group than in the control group (28.6±0.8 vs 19.3±0.2 mU/kg body weight/min respectively), and so was glucose uptake in skeletal muscle and the epididymal fat pad. Concordantly, insulin receptor (IR), IR substrate type 1 (IRS1), IRS2, and protein kinase B (AKT) phosphorylation in adipose tissue and skeletal muscle was improved by citrate ICV treatment. Moreover, the treatment with citrate for 7 days promoted body weight loss and decreased the adipose tissue. Our results suggest that citrate and glucose may serve as signals of energy and nutrient availability to hypothalamic cells.

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Maria H M Lima, Lílian C Souza, Luciana C Caperuto, Estela Bevilacqua, Alessandra L Gasparetti, Ricardo Zanuto, Mario J A Saad and Carla R O Carvalho

Polycystic ovary syndrome (PCOS) manifests as chronic anovulation, ovarian hyperandrogenism, and follicular cysts, which are amplified by insulin as well as the inability of the hormone to stimulate glucose uptake in classic target tissues such as muscle and fat. In the present study, we evaluated the regulation of the insulin-signaling pathways by using immunoprecipitation and immunoblotting in whole extracts of ovaries from non-pregnant human chorionic gonadotropin (hCG)-treated rats, hyperinsulinemic-induced rats and hyperinsulinemic-induced rats, treated with hCG for 22 consecutive days. There were increased associations of insulin receptor substrate (IRS)-1 and IRS-2 with phosphatidylinositol (PI) 3-kinase, followed by enhanced protein kinase B (Akt) serine and threonine phosphorylation, in the ovaries of rats that were treated with hCG, either alone or with insulin. In contrast, the skeletal muscle demonstrated a reduced IRS-1/PI 3-kinase/Akt pathway in hyperinsulinemic-induced rats. These intracellular modifications were accompanied by follicular cysts, detected by optical microscopy, and increased androstenedione serum levels. In summary, our data show that chronic treatment with hCG or hCG plus insulin can induce changes in ovaries that simulate PCOS. In these situations, an increase in the insulin-induced IRS/PI 3-kinase/Akt pathway occurs in the ovary, suggesting that the activation of this pathway may have a role in the development of PCOS.